Gene therapy and nucleic acid immunisation are promising approaches for the treatment and prevention of both acquired and inherited diseases. These techniques provide for the transfer of a desired nucleic acid into a subject with subsequent in vivo expression. Transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host. Alternatively, the nucleic acid can be administered in vivo directly to the recipient.
Each of these techniques requires efficient expression of the nucleic acid in the transfected cell, to provide a sufficient amount of the therapeutic or antigenic gene product. Several factors are known to affect the levels of expression obtained, including transfection efficiency, and the efficiency with which the gene or sequence of interest is transcribed and the mRNA translated.
A number of expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. Commonly used promoters for mammalian cell expression systems include the SV40 early promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (Chapman et al (1991) Nucl. Acids Res. 19:3979-3986), the mouse mammary tumour virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP) and the herpes simplex virus (HSV) promoter, among others. Nonviral promoters, such as a promoter derived from the murine metallothionein gene are also commonly used.
Expression systems often include transcriptional modulator elements, referred to as “enhancers”. Enhancers are broadly defined as a cis-acting agent, which when operably linked to a promoter/gene sequence, will increase transcription of that gene sequence. Enhancers can function from positions that are much further away from a sequence of interest than other expression control elements (e.g. promoters); and can operate when positioned in either orientation relative to the sequence of interest (Banerji et al. (1981) Cell 27:299-308, deVilleirs et al. (1981) Nucl. Acids Res 9: 6251-6264). Enhancers have been identified from a number of viral sources, including polyoma virus, BK virus, cytomegalovirus (CMV), adenovirus, simian virus 40 (SV40), Moloney sarcoma virus, bovine papilloma virus and Rous sarcoma virus (deVilleirs et al supra, Rosenthal et al. (1983) Science 222:749-755, Hearing et al (1983) Cell 33:695-703, Weeks et al. (1983) Mol. Cell. Biol. 3:1222-1234, Levinson et al. (1.982) Nature 295:568-572, and Luciw et al. (1983) Cell 33: 705-716).
A number of expression systems for nucleic acid immunisation and gene therapy make use of the hCMV immediate early promoter. See eg U.S. Pat. Nos. 5,168,062 and 5,385,839 to Stinski, and EP Patent Specification 0323997 B1. Expression vectors using the hCMV immediate early promoter include for example, pWRG7128 (Roy et al, Vaccine 19, 764-778, 2001), and pBC12/CMV and pJW4303 which are mentioned in WO 95/20660. Chapman et al (1991) report reduced levels of expression from the hCMV immediate early promoter in the absence of hCMV Intron A.